Therapeutic targets for adrenocortical carcinoma

ABSTRACT

This invention identifies and provides a recurrent translocation t(4;8) (p16.2; p23.1) associated with Adrenocortical Carcinoma, and diagnostic methods using the translocation by FISH hybridization or PCR based assays.

CROSS REFERENCE

This application is related to and claims the priority benefit of U.S.provisional application 61/542,126, filed on Sep. 30, 2011, theteachings and content of which are incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to a therapeutic target for AdrenocorticalCarcinoma (ACC). Further, it relates to methods of using a genomicapproach to identify a candidate therapeutic target, and methods ofusing the target for diagnosis or prognosis testing and drugdevelopment.

BACKGROUND OF THE INVENTION

Adrenocortical carcinoma (ACC) is an aggressive malignancy of theadrenal cortex with a poor 5-year survival rate of 10-20%. Many ACCpatients have no symptoms until their tumors reach a large size.Currently, there is no reasonably sensitive or specific way todistinguish ACC from the much more common benign adenomas. Right now,diagnosis is made on the basis of tumor size and histopathologicalfeatures that can be summarized by the Weiss score. Weiss scores of 0 or1 are considered benign, 2 and 3 are ambiguous, and 4 or larger arecancerous. Histological diagnosis of ACC is difficult to make, whichmakes treatment decisions complicated. It is evident from geneexpression profiling studies that some subset of tumors classified asadenomas turn out to be transcriptionally more similar to ACC. Localrecurrence is not sufficient to establish the diagnosis of ACC either.Metastatic disease or invasion into a contiguous structure is the onlyabsolute indicator of malignant disease in masses of the adrenal cortex.In addition to the challenges to the cancer diagnosis, there is a lackof clinical studies to guide therapy. Current therapy is oftenineffective and may also be associated with intolerable side effects.Outside of CT or PET scanning, there is no way to evaluate whethertreatment is working in reducing tumor burden. Therefore, there is aneed to develop a test based on a molecular abnormality in ACC that canbe used for identification and quantification of circulating tumor cellsat an early stage of the cancer.

SUMMARY OF THE INVENTION

One aspect of the present invention provides a confirmatory diagnosticmethod for Adrenocortical carcinoma (ACC). The general method comprisesthe steps of obtaining a sample from a subject suspected to haveadrenocortical carcinoma; and detecting a translocation abnormalityt(4;8) (p16.2; p23.1) in cells from the sample, wherein the presence ofthe translocation in more than about 15% of cells scored as positiveconfirms the diagnosis of ACC.

In some embodiments of the invention, the step of detecting t(4;8)(p16.2; p23.1) in the general method comprises karyotyping interphasechromosomes using fluorescent in situ (FISH) procedures, includingmulticolor-FISH (mFISH), split-signal FISH (ssFISH), Fusion signal FISH(fs), and any derivative procedure thereof, wherein the procedurecomprises one or more probes having sequence complementary to a sequencespecific to t(4;8) (p16.2; p23.1). In another embodiment of theinvention, the step of detecting t(4;8) (p16.2; p23.1) in the generalmethod comprises analyzing nucleic acid sequences specific to t(4;8)(p16.2; p23.1) using PCR, hybridization, sequencing, or any combinationthereof. In yet another embodiment of the invention, the step ofdetecting t(4;8) (p16.2; p23.1) in the general method comprisesanalyzing the expression of one or more genes disrupted by t(4;8)(p16.2; p23.1) using PCR, hybridization, sequencing, or any combinationthereof.

Another aspect of the invention provides a confirmatory diagnosticmethod for adrenocortical carcinoma (ACC). The method comprisesobtaining a sample from a subject suspected to have adrenocorticalcarcinoma; and detecting a translocation abnormality t(4;8) (p16.2;p23.1) using karyotyping interphase chromosome by a fluorescent in situ(FISH) procedure wherein the procedure comprises: (i) probes forChromosome 4 comprising one or more Bacterial Artificial Chromosomes(BACs) selected from the group consisting of: RP11-959C10, CTD2255016and RP11-803H22; and (ii) probes for Chromosome 8 comprising one or moreBACs selected from the group consisting of: RP11-54I15, RP11-1130G3, andCTD-2045B18. In this method, the presence of the translocation in a cellof the sample is signified by a distance between the chromosome 4 probessignal and the chromosome 8 probe signal that is less than a signal'swidth apart; and the presence of the translocation in more than about15% of cells scored as having the translocation confirms the diagnosisof ACC.

Additional aspect of this invention comprises a confirmatory diagnosticmethod for adrenocortical carcinoma (ACC), which comprises obtaining asample from a subject suspected to have adrenocortical carcinoma; anddetecting a translocation abnormality t(4;8) (p16.2; p23.1) using a PCRassay comprising probes that comprise sequences selected from the groupconsisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4,SEQ ID NO. 5, SEQ ID NO.6, SEQ NO. 7 and SEQ NO. 8.

Another aspect of the present invention provides a transgenic animal,comprising t(4;8) (p16.2; p23.1). For example, the transgenic animal maybe a mouse.

Yet another aspect of the present invention provides a method foridentifying an agent that reduces ACC cell viability. The general methodcomprises the steps of contacting the cancer cell with an agent, whereinthe cancer cell comprises t(4;8) (p16.2; p23.1); and testing one or morecancer cell responses to the agent, wherein the cancer cell response isselected from the group consisting of tumor cell count, metastasis,apoptosis, wherein a lower level of drug target activity, cancer cellcount, or metastasis indicates that the agent is a therapeutic agentagainst ACC; wherein the cancer cell response is compared relative to acontrol sample. In the general method, the agent is preferably apharmaceutically active ingredient or pharmaceutically acceptable saltthereof, a drug, a toxin, a chemical, a small organic molecule, a largemolecule or peptide, or an antibody.

Other aspects and iterations of the invention are described in moredetail below.

REFERENCE TO COLOR FIGURES

The application file contains at least one figure executed in color.Copies of this patent application publication with color figures will beprovided by the Office upon request and payment of the necessary fee.

DESCRIPTION OF THE FIGURES

FIG. 1 depicts the IGV screen shots of the region of interest from ACC132. Top panel highlights chromosome 4 and the bottom panel shows thecorresponding jump to chromosome 8;

FIG. 2 depicts the fluorescent in situ hybridization (FISH) withRP11-959C10 (red) and RP11-54115 (green). There is evidence forco-localization with yellow signals (arrows); and

FIG. 3 depicts the PCR amplification of putative translocationbreakpoints. The der(4) product is in lanes 2-4 and 6-8, and the der(8)product is in lanes 10-12 and 14-16. The ladder is a 100 bp ladder.Amplification of ˜700 bp fragment was seen in the der(4)t(4;8)(p16.2;p23.1) PCR. The predicted 517 bp product of theder(8)t(4;8)(p16.2;023.1) was not observed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a translocation associated withAdrenocortical Carcinoma (ACC) and a method for ACC diagnosis using thetranslocation. The invention also provides a transgenic animalcomprising the translocation, and a method of identifying ACCtherapeutics using the translocation disclosed herein.

Translocations are chromosomal abnormalities which occur whenchromosomes break and the fragments of the chromosomes rejoin to otherchromosomes. Translocations are often detected on cytogenetics or akaryotype of affected cells. The two principal molecular consequences oftranslocations are the activation of proto-oncogenes and the creation ofnovel fusion genes. Translocations are rearrangements between twochromosomes where portions of different chromosomes are exchanged. Atranslocation is classified as recurrent when it occurs in more than onesample of the same type tumor from different patients.

The present invention provides a recurrent translocation, t(4;8) (p16.2;p23.1), associated with ACC. t(4;8) (p16.2; p23.1) has break pointsaround chr4:4090373—specifically within the short arm of the chromosome4 region 1 band 6 sub-band 2, and chr8:6993890—specifically within theshort arm of the chromosome 8 region 2 band 3 sub-band 1.

Previous work in the field of genomic profiling has used eitherG-banding or CGH (Comparative genomic hybridization) method. G-bandingis a technique used in cytogenetics to produce a visible karyotype bystaining condensed chromosomes. G-banding suffers from both a lack ofresolution (10 mega base) and ambiguity because banding is not 100%specific for chromosomal regions. Therefore, using G-banding alone wouldbe inadequate for identification of structural rearrangements. CGHinvolves hybridizing differentially labeled DNA from tumor and a normalcontrol to metaphase chromosomes. The level of resolution obtainabledepends on the length of the metaphase chromosomes and has a limit of 10Mb as well. Therefore, CGH cannot detect structural rearrangements liketranslocations. Further, the prevalent genomic duplication, althoughlikely facilitating DNA rearrangement, prevents absolute determinationof the rearrangement by analysis of the sequence alone. Thus, neither ofthese techniques can be readily used to detect the discoveredabnormality. In one embodiment of the invention, the combination ofspectral karyotyping and DNA sequencing of the entire genome of the ACCcell lines, allowing for a more precise identification of structuralrearrangements, was used to detect the recurrent translocation t(4;8)(p16.2; p23.1). The ACC cell lines that may be used for spectralkaryotyping are selected from a group consisting of SW-13, H295, H295R,and any derivatives thereof. In one embodiment, the ACC cell lines usedfor spectral karyotyping are SW-13 and H295R. The DNA sequencing ispreferably in-depth, whole genome sequencing in the regions implicatedby cell line spectral karyotyping in one or more ACC tumors.

In another aspect of the present invention, a recurrent translocationassociated with ACC tumors can be used to develop a diagnostic test forACC. The presence of a target recurrent translocation may be detected bymethods that are PCR-based, hybridization-based, sequencing-based, orany combination of the above methods or derivatives thereof. Thedetection may be through various methods including, but not limited to,PCR-based methods including real-time PCR, quantitative PCR, andquantitative real time PCR.

In one embodiment, the diagnostic test is a FISH-based molecularcytogenetics assay. The technique generally entails preparing a sample,labeling probes, denaturing target chromosomes and the probe,hybridizing the probe to the target sequence, and detecting a signal.

In one embodiment, the test is a multicolor-FISH technique (m-FISH ormultiplex FISH) such that each separate normal chromosome is stained bya separate color. In another embodiment, split-signal FISH (ssFISH)detects changes in chromosomal structure by using two probes, each ofwhich is labeled by a different detectable label. Preferably, thedetectable labels should be distinguishable from one another. Each probebinds to the chromosome on either side of a suspected breakpoint in thechromosome. In a normal chromosome, the two probes will be proximalenough to each other such that the combined signal of their differentlabels forms a signal that is different from each label alone. Thus, anormal chromosomal sample will contain only the combined or fused signalof the two probes on the sister chromosomes. In an abnormal sample,where one sister chromosome has broken at the suspected break point, thefused signal will remain on the normal sister chromosome. On the brokenchromosome, one probe migrates to a different chromosome, where theindividual signal of that probe becomes apparent. The other individualprobe remains on the split chromosome and, because it is no longerproximal to the other probe, emits its individual signal as well. Insum, because of the break in chromosomal structure, the two probes areno longer juxtaposed, allowing the fused signal they form together tosplit into the individual signals for each probe.

Fusion-signal FISH is similar to ssFISH in that two probes with twodifferent, distinguishable labels are used such that proximity of thetwo labels produces a new fused signal. The two methods differ in thatfor fusion-signal FISH, the two probes bind to two different chromosomalpairs at locations that are suspected to become proximal to each otheras a result of a chromosomal rearrangement. Thus, in a normal sample,only signals from the individual probes are present and no fused signalappears. In an abnormal sample, where a piece of one chromosome hasattached to another chromosome, the normal chromosomes in each of thetwo pairs involved will still emit each of the individual probe signals.In the abnormal chromosome, in which the probes are now proximal due tothe translocation, the fused signal appears.

Alternatively, a diagnostic test in another embodiment is a PCR-basedmethod detecting the presence of the translocation. In addition, therecurrent translocation t(4;8) (p16.2; p23.1) involves two genes ofunknown function (BC042823 (uc003gho.2, IMAGE:5275587) on chromosome 4and BC030294 (IMAGE:5396854) on chromosome 8 to create a fusion gene.Translocation may result in a disrupted gene or a gene fusion. ThereforePCR-based methods can be used to detect those events as well. Thedisruption of a gene may lead to an increased or decreased expressionlevel, and/or altered expression specificity. As used herein, a genefusion refers to an accidental joining of the DNA of two genes. Genefusions may give rise to hybrid proteins or the misregulation of thetranscription of one gene due to the juxtaposition of cis regulatoryelements (e.g., enhancers or promoters) of another gene. In oneembodiment of the diagnostic test based on t(4;8) (p16.2; p23.1), thetest is to detect the expression level of one or both of the genes ifthe gene is disrupted due to the translocation. In another embodiment,the test is to detect the presence of a hybrid nucleic acid or protein.

In calculating the expression of a target gene in relation to anadequate reference gene, various mathematical models are established.Calculations are based on the comparison of the distinct cycledetermined by various methods, e.g., crossing points (CP) and cyclethreshold values (Ct) at a constant level of fluorescence; or CPacquisition according to established mathematic algorithms.

The algorithm for Ct values in RT-PCR calculates the cycle at which eachPCR amplification reaches a significant threshold. The calculated Ctvalue is proportional to the number of target copies present in thesample, and the Ct value is a precise quantitative measurement of thecopies of the target found in any sample. In other words, Ct valuesrepresent the presence of the respective target that the primer sets aredesigned to recognize. If the target is missing in a sample, thereshould be no amplification in the RT-PCR reaction. Alternatively, the Cpvalue may be utilized. Cp value represents the point in the cycle atwhich the increase of fluorescence is highest and where the logarithmicphase of a PCR reaction begins. For example, the LightCycler® 480Software (Roche Applied Science, Penzberg, Germany) calculates thesecond derivatives of entire amplification curves and determines wherethis value is at its maximum. By using the second-derivative algorithm,data obtained are more reliable and reproducible, even if fluorescenceis relatively low. The expression of the biomarker or target in the testsubject may be 1,000,000×, 100,000×, 10,000×, 1000×, 100×, 10×, 5×, 2×,1×, 0.5×, 0.1×, 0.01×, 0.001×, 0.0001×, 0.00001×, 0.000001×, or0.0000001× of the predetermined level indicating the presence or absenceof a cellular or physiological characteristic. The predetermined levelof expression may be derived from a single control sample or a set ofcontrol samples.

The various and non-limiting embodiments as described herein maycomprise one or more probes and/or primers. Generally, the probe orprimer contains a sequence complementary to a sequence specific to theregion of the translocation, or specifically to the genes in the regionof translocation. A homologous sequence having less than 60% 70%, 80%,90%, 95%, 99% or 100% identity to the identified gene sequence may alsobe used for probe or primer design if it is capable of binding to itscomplementary sequence of the desired target sequence in the region oftranslocation under stringent condition. “Homologous” refers to anyprobe which can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions ranging from lowto high levels of stringency.

Low stringency conditions, when used in reference to nucleic acidhybridization, comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.1% SDS, 5×Denhardt's reagent [50×Denhardt's contains per 500ml: 5 g Ficoll (Type 400, Pfizer, New York, N.Y.), 5 g BSA (Fraction V;Sigma-Aldrich, St. Louis, Mo.)] and 100 μg/ml denatured salmon sperm DNAfollowed by washing in a solution comprising 5×SSPE, 0.1% SDS at 42° C.when a probe of about 500 nucleotides in length is employed.

High stringency conditions, when used in reference to nucleic acidhybridization, comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.5% SDS, 5×Denhardt's reagent and 100 μg/ml denatured salmonsperm DNA followed by washing in a solution comprising 0.1×SSPE, 1.0%SDS at 42° C. when a probe of about 500 nucleotides in length isemployed.

It is well known that numerous equivalent conditions may be employed tocomprise low stringency conditions; factors such as the length andnature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,etc.) and the concentration of the salts and other components (forexample, the presence or absence of formamide, dextran sulfate,polyethylene glycol) are considered and the hybridization solution maybe varied to generate conditions of low stringency hybridizationdifferent from, but equivalent to, the above listed conditions. Inaddition, the art knows conditions that promote hybridization underconditions of high stringency (for example, increasing the temperatureof the hybridization and/or wash steps, the use of formamide in thehybridization solution, etc.).

Generally, nucleic acid based probes and primers are complementary to asequence within the target DNA sequence region. Probes may include oneor more labels. A label may be any substance capable of aiding amachine, detector, sensor, device, or enhanced or unenhanced human eyefrom differentiating a labeled composition from an unlabeledcomposition. Examples of labels include, but are not limited to: aradioactive isotope or chelate thereof, dyes (fluorescent ornonfluorescent,) stains, enzymes, or nonradioactive metals. Specificexamples include, but are not limited to: fluorescein, biotin,digoxigenin, alkaline phosphatese, biotin, streptavidin, ₃H, ₁₄C, ₃₂P,₃₅S, or any other compound capable of emitting radiation, rhodamine,4-(4_-dimethylaminophenylazo) benzoic acid (“Dabcyl”),4-(4_-dimethylamino-phenylazo)sulfonic acid (sulfonyl chloride)(“Dabsyl”), 5-((2-aminoethyl)-amino)-naphtalene-1-sulfonic acid(“EDANS”), psoralene derivatives, haptens, cyanines, acridines,fluorescent rhodol derivatives, cholesterol derivatives,ethylenediaminetetraaceticacid (“EDTA”) and derivatives thereof, or anyother compound that may be differentially detected. The label may alsoinclude one or more fluorescent dyes optimized for use in genotyping.Examples of such dyes include, but are not limited to: CAL-Fluor Red610, CAL-Fluor Orange 560, dR110, 5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET,dTAMRA, TAMRA, NED, dROX, PET, BHQ+, Gold540, Cy3, Cy5, LIZ, and TexasRed.

The detection of fusion protein due to the translocation may be throughnovel epitopes recognized by polyclonal and/or monoclonal antibodiesused in ELISA, immunoblotting, flow cytometric, immunohistochemical, andother mutant protein detection strategies (as described in Wong et al.,Cancer Res., 46: 6029-6033, 1986; Luwor et al., Cancer Res., 61:5355-5361, 2001; Mishima et al., Cancer Res., 61: 5349-5354, 2001; Ijazet al., J. Med. Virol., 63: 210-216, 2001, the teachings and content ofwhich are incorporated by reference herein). The term “antibody” is usedherein in the broadest sense and refers generally to a molecule thatcontains at least one antigen binding site that immunospecifically bindsto a particular antigen target of interest. The term antibody thusincludes, but is not limited to, native antibodies and variants thereof,fragments of native antibodies and variants thereof, peptibodies andvariants thereof, and antibody mimetics that mimic the structure and/orfunction of an antibody or a specified fragment or portion thereof,including single chain antibodies and fragments thereof. The term thusincludes full length antibodies and/or their variants as well asimmunologically active fragments thereof, thus encompassing antibodyfragments capable of binding to a biological molecule (such as anantigen or receptor) or portions thereof, including but not limited toFab, Fab′, F(ab′)2, facb, pFc′, Fd, Fv or scFv (See, e.g., CURRENTPROTOCOLS IN IMMUNOLOGY, (Colligan et al., eds., John Wiley & Sons,Inc., NY, 1994-2001)).

The sample in this general diagnostic method is preferably a biologicalsample from a subject. The term “sample” or “biological sample” is usedin its broadest sense. Depending upon the embodiment of the invention,for example, a sample may comprise a bodily fluid including whole blood,serum, plasma, urine, saliva, cerebral spinal fluid, semen, vaginalfluid, pulmonary fluid, tears, perspiration, mucus, and the like; anextract from a cell, chromosome, organelle, or membrane isolated from acell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to asubstrate; a tissue; a tissue print; or any other material isolated inwhole or in part from a living subject. Such samples include, but arenot limited to, tissue isolated from primates, e.g., humans, or rodents,e.g., mice, and rats. Biological samples may also include sections oftissues such as biopsy and autopsy samples, and frozen sections takenfor histologic purposes such as blood, plasma, serum, sputum, stool,tears, mucus, hair, skin, and the like. Biological samples also includeexplants and primary and/or transformed cell cultures derived frompatient tissues. The term “subject” is used in its broadest sense. In apreferred embodiment, the subject is a mammal. Non-limiting examples ofmammals include humans, dogs, cats, horses, cows, sheep, goats, andpigs. Preferably, a subject includes any human or non-human mammal,including for example: a primate, cow, horse, pig, sheep, goat, dog,cat, or rodent, capable of developing cancer including human patientsthat are suspected of having cancer, that have been diagnosed withcancer, or that have a family history of cancer.

The probes and primer sequences to work with PCR, for example, include:der(4)t(4;8)_(—)4_F: ctcgccccacagtatcttatca (SEQ ID NO. 1);der(4)t(4;8)_(—)4_R: aaaaaCCAACCTAGGGCTGTC (SEQ ID NO. 2);der(8)t(4;8)(p16;p23)_Primer_F: gatgtgctaagagtcagcttgc (SEQ ID NO. 3)and der(8)t(4;8)(p16;p23)_Primer_R: tgggcaacagtgagactttgt (SEQ ID NO.4); der(4)t(4;8)_(—)4_(—)2_F: GACGGCCAGTACTTCTTTCATCTGTTTTGTGTTGG (SEQID NO. 5); der(4)t(4;8)_(—)4_(—)2_R: TATGACCATGAGGGATGGGATCTGGGTGATTA(SEQ ID NO. 6); der(8)t(4;8)_(—)8_(—)2_F:GACGGCCAGTTGACCTAGCCCCTCTCTTTCTC (SEQ ID NO. 7) andder(8)t(4;8)_(—)8_(—)2_R: TATGACCATGTGACCTAGCCCCTCTCTTTCT (SEQ ID NO.8). FISH probes for Chromosome 4 are BACs (Bacterial ArtificialChromosome): RP11-959C10, CTD2255016, RP11-803H22, RP11-351L3,RP11-357G3, and RP11-687G23. FISH probes for Chromosome 8 are BACs:RP11-54I15, RP11-1130G3, RP11-826L17, RP11-623J22 and CTD-2045B18.

The various embodiments of the test developed based on the ACCassociated recurrent translocation t(4;8) (p16.2; p23.1) may be used tomonitor disease progression or as a companion test for ACC therapeutics.

There are only a few widely available ACC cell lines, including SW13,H295 and H295R. ACC is rare in mice. Another aspect of the inventionprovides a cell line cell or a genetically modified animal comprisingthe ACC associated recurrent translocation t(4;8) (p16.2; p23.1). Inanother embodiment, the cell line cell or the genetically modifiedanimal comprises the disrupted gene or gene fusion in the region of therecurrent translocation t(4;8) (p16.2; p23.1). For example, such atransgenic mouse model may then serve as an animal model to evaluate newtherapies for the treatment of patients with ACC.

The development of novel pharmaceutical therapeutics relies on theidentification and validation of key regulators of a drug targets, whichmay be the disrupted, or the fusion gene involved in the region of theACC associated recurrent translocation t(4;8) (p16.2; p23.1).

Another aspect of the invention provides methods for identifying an ACCtherapeutic agent through a drug target based on the recurrenttranslocation as disclosed herein. The methods comprise contacting atest agent with a cell comprising a therapeutic target that is expressedin the cell. An agent that is an inhibitor of cancer condition may beidentified by determining the effect of a test agent on the expressionlevel of a target. In a particular example, a test agent thatdown-regulates the target expression as compared to the targetexpression in the absence of the test agent identifies that test agentas an inhibitor of a target.

Inhibitors of drug target gene or protein expression may be any agentincluding a pharmaceutically active ingredient or pharmaceuticallyacceptable salt thereof, a drug, a toxin, a chemical, a small organicmolecule, a large molecule or peptide, or an antibody. Large-moleculepharmaceuticals refer to pharmaceutical agents having a molecular weightgreater than about 1000 daltons, e.g. peptidic drugs, vaccines andhormones. The term “antibody” has the same definition as providedherein.

The screening or creation, identification, and selection of appropriateinhibitors of drug targets for ACC can be accomplished by a variety ofmethods. One approach is to use structural knowledge about the targetprotein to design a candidate molecule with which it will preciselyinteract. An example would be computer assisted molecular design. Asecond approach is to use combinatorial or other libraries of molecules,whereby a large library of molecules is screened for inhibitory effectwith regard to the target gene or protein expression, or ability toinhibit the transcriptional factor activity of the target protein. In afurther example, a panel of antibodies may be screened for ability toinhibit the target protein.

Cancer and precancer may be thought of as diseases that involveunregulated cell growth. Metastasis involves migration of tumor cellsaway from the site of the primary tumor, entry into the circulation, andproliferation at a new site. Cell growth involves a number of differentfactors. One factor is how rapidly cells proliferate, and anotherinvolves how rapidly cells die. Cells can die either by necrosis orapoptosis depending on the type of environmental stimuli. Cell motilityis yet another factor that influences tumor growth kinetics andmetastasis. Resolving which of the many aspects of cell growth a testagent affects can be important to the discovery of a relevantpharmaceutical therapy for ACC cancer cells. Screening assays based onthis technology can be combined with other tests to determine whichagents have growth inhibiting and pro-apoptotic activity in ACC cancercells.

Some embodiments provided herein involve determining the ability of agiven agent to inhibit the expression of the ACC drug targets. Testagents can be assessed for their probable ability to inhibit growth orotherwise alter the behavior of ACC cancer cells. Various cell lines canbe used and could be selected based on the tissue to be tested. Certaincell lines are well characterized, and are used, for instance, by theUnited States National Cancer Institute (NCl) in their screening programfor new anti-cancer drugs. Cell lines can also be constructed tooverexpress the ACC drug targets for screening inhibitory agents for ACCcancer cells. Significant tumor cell growth inhibition, greater thanabout 30% at a dose of 1001.1M or below, is further indicative that theagent is useful for treating the ACC. An IC₅₀ value may be determinedand used for comparative purposes. This value is the concentration ofdrug needed to inhibit tumor cell growth by 50% relative to the control.In some embodiments, the IC₅₀ value is less than 100 μM in order for theagent to be considered further for potential use for treating,ameliorating, or preventing tumor metastasis.

EXAMPLES

The following non-limiting examples are included to illustrate theinvention, and they are not intended to limit the scope of the claims.

Example 1 Whole Genome Sequencing on an ACC Tumor and Matched PeripheralBlood

Clinical Synopsis

A 51 year old female presented with abdominal fullness and pressureafter eating. A CT scan demonstrated a 9.5 cm left adrenal tumor. Shehad no excess adrenal hormone on functional testing. She underwent alaparoscopic left adrenalectomy with tumor capsule fracture. Three yearslater, she was found to have local recurrence by CT and was started onmitotane for about 6 months during which time the tumors continued togrow. She was then referred for the OSI-906 trial study, on which sheremained for 8 months. There was no evidence of distant disease, so thepatient was returned to the operating room for resection of the locallyrecurring intraperitoneal disease 4 years after presentation. Multipleareas of recurrent ACC were resected from the omentum, along the leftkidney, aorta, descending colon and the diaphragm. The largest focus ofcancer was 14.5 cm. The modified Weiss score of this sample was 4. Afterthis operation, she received adjuvant chemotherapy in the form of 4cycles of etoposide, cisplatin and doxorubicin. Six months aftersurgery, her CT showed no evidence of recurrence.

Sequencing:

DNA extracted from both tumor and blood was processed independently, andlibraries were paired-end sequenced on the Illumina HiSeq 2000 platform(Illumina, Inc., San Diego, Calif.).

Analysis:

Sequencing data was aligned to the reference genome, build hg18.Variations were predicted in the tumor and blood separately using twodifferent SNP calling algorithms: Varscan (Washington University, St.Louis, Mo.) and an internal caller (TGen, Phoenix, Ariz.). After SNPprediction, results were combined to identify variations called by bothalgorithms, submitted to PolyPhen2 to assess predicted functionalconsequence, and filtered to yield those SNPs most likely to representdeleterious, non-synonymous changes in coding regions of the genome inthe tumor specifically, as well as those shared by the tumor and blood(i.e., constitutional changes). Copy number variation was computed.Further analysis of structural variation included indels andtranslocations. ACC is not characterized by mutations in commonlyaltered cancer genes, and the same observation was obtained from the twoACC cell lines, SW-13 and H295, as well.

Example 2 The Translocation Discovered Using a Genomic Approach

The combination of spectral karyotyping and DNA sequencing of the entiregenome of the ACC cell lines allows more precise identification ofstructural rearrangements. The translocation was discovered by analyzingthe data from the first in-depth, whole genome sequencing of two ACCtumors in regions implicated by the spectral karyotyping of the two ACCcell lines, SW-13 and H295R. A recurrent translocation,t(4;8)(p16.2;p23.1) with break points around chr4:4090373 andchr8:6993890.

Example 3 The Translocation Validation

The translocation was validated by fluorescent in situ (FISH) in an ACCcell line from a different tumor using BACs spanning the putativebreakpoints. Two BACs spanning the break points: Chr 4: RP11-959C10(TAMRA labeled, red) Chr 8: RP11-54115 (FITC labeled, green) werediscovered. The two BACs were labeled fluorescently and used to FISH inACC 140-1, a cell line derived from another ACC tumor. Theoretically,the red and green probes would hybridize to the breakpoint and appear toco-localize, identifying the existence of the translocation, and thusgive a yellow signal. In comparison, normal chromosomes 4 and 8 withouttranslocation would have green or red signals, respectively. As shown inFIG. 2, there was evidence of co-localization.

The presence of the translocation was also validated using PCR toamplify the putative breakpoints. The putative translocation productshave distinct sizes from the other PCR products and can be resolved bygel electrophoresis. FIG. 3 shows the presence of a ˜700 bp fragment inH295R.

Example 4 Prevalence and Specificity of the Translocation in ACC

The recurrent translocation, t(4;8)(p16.2;p23.1), with break pointsaround chr4:4090373 and chr8:6993890, was shown to lie near to oneanother in normal cells. This information was utilized in setting ascoring criteria for the FISH protocol in detecting this t(4;8)translocation in both metaphase spreads and interphase nuclei. In theFISH protocol, an interphase cell was scored as positive if the distancebetween the chromosome 4 probe signal and the chromosome 8 probe signalis less than a signal's width apart. Interphase FISH to normal bloodwere carried out to evaluate t(4;8) translocation in both interphasecells and metaphase spreads as control, which demonstrated a backgroundrate of 10 false positives for every 100 interphase cells scored. Therewere no metaphase spreads found positive for the t(4;8) translocation innormal blood. However, in the evaluation of metaphase spreads in thecells from ACCs that carry the translocation, a portion of the metaphasespreads were found to be positive for the t(4;8) translocation. The datasuggested that samples having more than about 15% of cells scored aspositive in t(4;8) translocation can be reliably called positive. Table1 lists the ranges of positive scores from the tumor samples comprisingnuclei from fresh frozen tissues.

TABLE 1 T(4; 8) Scoring in Tumor Samples Sample Type ACC Adrenal Non-ACCNon-ACC tumor Adenoma Malignant Tumor Benign Tumors Number of 5 2 3 2Tumors Assayed Range of 18-30 11-17 25-31 15-16 positive cells/100 cellsscored

Table 1 demonstrates that 1) ACC tumor samples usually have 20-30%positive cells and 2) other malignant tumors are also scoring aspositive in the range of 20-30% cells. This suggests that although thetranslocation is not ACC specific, it is specific for malignant tumors,much like mutations in p53 are not specific for a particular type ofcancer but can be found in 50% of all malignant tumors.

Various cell lines were also scored for the recurrent translocation,t(4;8)(p16.2;p23.1). Those cell lines included: HCT116 (Human ColorectalCarcinoma cell line); ACC167 (Human Adrenocortical Carcinoma); H295Rcell line (Human Adrenocortical Carcinoma); SW-13 (Human AdrenocorticalCarcinoma); ACC140-1 (Human Adrenocortical Carcinoma). Table 2 lists theranges of positive scores from these cell lines.

TABLE 2 T(4; 8) Scoring in Cell lines Cell Type HCT116 ACC167 H295RSW-13 ACC140-1 Range of 2/100 11/100 10/100 31/100 18/100 positivecells/100 cells scored

Example 5 t(4;8) Translocation as a Therapeutic Target of ACC

Tumor samples, both benign and malignant as well as ACC and non-ACC wereassayed to determine exactly what the prevalence is for differentdiagnoses. In addition, the t(4;8) translocation was not found in allACC cells, and thus, it is a necessary but not sufficient event in thedevelopment of ACC. The fact that t(4;8) translocation is maintained inthe tumor and not lost completely suggests that this translocationrepresents a novel therapeutic target. The RNA-sequencing data of threeACC tumors provided such evidence that altered transcription from thisregion were discovered. PCR assay was designed to detect the predictedproducts and to show consistent transcription alteration from all threetumors. Part of the products showed homology to LOC100506990, which ispredicted to be uncharacterized LOC100506990 gene (GeneBank Access No:NR_(—)040091.1).

Example 6 Prevalence and Specificity of the Translocation in ACC

An interphase FISH screening with the probe sets comprising Chromosome 4probe (RP11-959C10, CTD2255016, RP11-803H22, RP11-351L3, RP11-357G3, orRP11-687G23) and Chromosome 8 probe (RP11-54I15, RP11-1130G3,RP11-826L17, RP11-623J22 or CTD-2045B18) combinations thereof may beused to screen tumors, both benign and malignant, in an in-house adrenaltumor repository (TGen, Phoenix, Ariz.). Both fusion and split-apartFISH designs are possible with these probes. Probes were labeled eitherred or green, and hybridized either to nuclei dissociated from frozentissue or to FFPE tissues. 100-300 nuclei were then scored for eitherco-localization (in the case of a fusion design) or dissociation (incase of a split-apart design) and compared to the pattern observed onnormal tissue.

Example 7 Functional Characterization of the Product of theTranslocation

siRNA may be designed to target the two genes involved in thetranslocation. The translocation product selected will be a knockdown orknock-out using the siRNA. The capability of adrenocortical celltransforming and the condition for transformation is then determined.The phenotypes of the ACC cells such as apoptosis, autophagy, decreasedproliferation, decreased migration, inhibition of cell signalingpathways, etc. may be analyzed to facilitate ACC therapeuticdevelopment.

1. A confirmatory diagnostic method for Adrenocortical carcinoma (ACC),comprising obtaining a sample from a subject suspected to haveadrenocortical carcinoma; and detecting a translocation abnormalityt(4;8) (p16.2; p23.1) in cells from the sample, wherein the presence ofthe translocation in more than about 15% of cells scored as positiveconfirms the diagnosis of ACC.
 2. The method of claim 1, whereindetecting t(4;8) (p16.2; p23.1) comprises karyotyping interphasechromosomes using fluorescent in situ (FISH) procedure includingmulticolor-FISH (mFISH), split-signal FISH (ssFISH), Fusion signal FISH(fs) and any derivative procedure thereof, wherein the procedurecomprises hybridizing one or more probes having a sequence complementaryto a sequence specific to t(4;8) (p16.2; p23.1).
 3. The method of claim1, wherein detecting t(4;8) (p16.2; p23.1) comprises analyzing nucleicacid sequences specific to t(4;8) (p16.2; p23.1) using PCR,hybridization, sequencing or any combination thereof.
 4. The method ofclaim 1, wherein detecting t(4;8) (p16.2; p23.1) comprises analyzing theexpression of one or more genes disrupted by t(4;8) (p16.2; p23.1) usingassays selected from the group consisting of PCR, hybridization,sequencing and any combination thereof.
 5. The method of claim 2,wherein probes having sequence complementary to a sequence specific tot(4;8) (p16.2; p23.1) comprising: a) one or more probes for Chromosome 4comprising a Bacterial Artificial Chromosome (BAC) selected from thegroup consisting of: RP11-959C10, CTD2255016, RP11-803H22, RP11-351L3,RP11-357G3, and RP11-687G23; and b) one or more probes for Chromosome 8comprising a BAC selected from the group consisting of: RP11-54I15,RP11-1130G3, RP11-826L17, RP11-623J22 and CTD-2045B18. wherein the probefor Chromosome 4 and the probe for Chromosome 8 are labeled differently;wherein the presence of a translocation in a cell of the sample issignified when the distance between the signal from a chromosome 4 probeand the signal from a chromosome 8 probe is less than a signal's widthapart.
 6. The method of claim 3, wherein the PCR assay comprises probescomprising sequences selected from the group consisting of SEQ ID NO. 1,SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO.6, SEQ ID NO. 7 and SEQ ID NO. 8; wherein SEQ ID NO. 1 and SEQ ID NO.2are a pair of forward and reverse primers; wherein SEQ ID NO. 3 and SEQID NO. 4 wherein SEQ ID NO. 5 and SEQ ID NO. 6 are a pair of forward andreverse primers; and wherein SEQ ID NO. 7 and SEQ ID NO. 8 are a pair offorward and reverse primers.
 7. A confirmatory diagnostic method foradrenocortical carcinoma (ACC), comprising a) obtaining a sample from asubject suspected to have adrenocortical carcinoma; and b) detecting atranslocation abnormality t(4;8) (p16.2; p23.1) using karyotypinginterphase chromosome by a fluorescent in situ (FISH) procedure whereinthe procedure comprises: i) probes for Chromosome 4 comprising one ormore Bacterial Artificial Chromosomes (BACs) selected from the groupconsisting of: RP11-959C10, CTD2255016 and RP11-803H22; and ii) probesfor Chromosome 8 comprising one or more BACs selected from the groupconsisting of: RP11-54I15, RP11-1130G3, and CTD-2045B18. wherein thepresence of the translocation in a cell of the sample is signified by adistance between the chromosome 4 probes signal and the chromosome 8probe signal that is less than a signal's width apart; wherein thepresence of the translocation in more than about 15% of cells scored ashaving the translocation confirms the diagnosis of ACC.
 8. Aconfirmatory diagnostic method for adrenocortical carcinoma (ACC),comprising a) obtaining a sample from a subject suspected to haveadrenocortical carcinoma; and b) detecting a translocation abnormalityt(4;8) (p16.2; p23.1) using a PCR assay comprising probes that comprisesequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO.6, SEQ NO. 7 andSEQ NO.
 8. 9. The method of claim 8, wherein detecting the translocationabnormality t(4;8) (p16.2; p23.1) using a PCR assay comprises the stepsof: adding a forward primer that includes SEQ ID NO. 1, SEQ ID NO. 3,SEQ ID NO. 5 or SEQ ID NO. 7 to a first mixture, wherein the firstmixture comprises a nucleic acid isolated from the sample; adding areverse primer that includes SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6,or SEQ ID NO.8 to the first mixture; and subjecting the mixture toconditions that allow nucleic acid amplification; wherein forward primerSEQ ID NO.1 and reverse primer SEQ ID NO. 2 allow nucleic acidamplification in the mixture; wherein forward primer SEQ ID NO. 3 andreverse primer SEQ ID NO. 4 allow nucleic acid amplification; whereinforward primer SEQ ID NO. 5 and reverse primer SEQ ID NO. 6 allownucleic acid amplification; and wherein forward primer SEQ ID NO. 7 andreverse primer SEQ ID NO. 8 allow nucleic acid amplification; anddetecting the presence of the translocation on the basis of a result ofthe nucleic acid amplification.
 10. The method of claim 9, furthercomprising the step of adding an oligonucleotide probe to the mixture.11. The method of claim 10, wherein the oligonucleotide probe comprisesa label.
 12. The method of claim 11, wherein the label is selected froma radioactive isotope or chelate thereof, fluorescent dyes,nonfluorescent dyes, stains, enzymes, and nonradioactive metals.
 13. Themethod of claim 12, wherein the fluorescent label is selected from thegroup consisting of CAL-Fluor Red 610, CAL-Fluor Orange 560, dR110,5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET, dTAMRA, TAMRA, NED, dROX, PET,BHQ+, Gold540, Cy3, Cy5, LIZ, and Texas Red.
 14. The method of claim 9,wherein the result comprises a Ct value.
 15. The method of claim 9,wherein the result comprises a Cp value.